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Characteristics of Pos19 – A Small Coding RNA in the Oxidative Stress Response of Rhodobacter sphaeroides

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ABSTRACT

The phototrophic bacterium Rhodobacter sphaeroides induces several small RNAs (sRNAs) when singlet oxygen (1O2) levels are elevated, a situation also referred to as photo-oxidative stress. An RNA-seq study identified the RSs0019 sRNA, which is renamed Pos19 (photo-oxidative stress induced sRNA 19). Pos19 is part of the RpoE regulon and consequently induced upon 1O2 and peroxide stress. The 219 nt long Pos19 transcript contains a small open reading frame (sORF) of 150 nt, which is translated in vivo. Over-expression of Pos19 results in reduced mRNA levels for several genes, of which numerous are involved in sulfur metabolism. The negative effect on the potential targets is maintained even when translation of the sORF is abolished, arguing that regulation is entailed by the sRNA itself. Reporter studies further revealed that regulation of the most affected mRNA, namely RSP_0557, by Pos19 is Hfq-dependent. Direct binding of Pos19 to Hfq was shown by co-immunoprecipitation. Physiological experiments indicated Pos19 to be involved in the balance of glutathione biosynthesis. Moreover, a lack of Pos19 leads to elevated reactive oxygen species levels. Taken together our data identify the sRNA Pos19 as a coding sRNA with a distinct expression pattern and potential role under oxidative stress in the phototrophic bacterium R. sphaeroides.

No MeSH data available.


Regulation of RSP_0557 by Pos19 depends on Hfq.(A) Results from in vivo reporter studies. Gene fragments containing the first codons and part of the upstream region were translationally fused to the lacZ gene on plasmid pPHU235. The corresponding reporter plasmids were transferred to control (pBBR1) and over-expression strains of Pos19 (pPos19) in wild-type (wt 2.4.1) and hfq deletion mutant (Δhfq) backgrounds. Cultures were stressed with 1O2 for 60 min and samples subjected to β-galactosidase assays. Bars indicate the relative β-galactosidase activity as calculated from Miller units (with the pBBR1 control set to 100% for each construct). Results represent the mean from three independent experiments with technical duplicates and error bars reflect the standard deviation. (B) Northern blot results from Hfq coIP experiments. Control plasmid pBBR1 and over-expression plasmid pPos19 were transferred to strains either expressing wild-type Hfq (wt hfq+) or 3xFLAG-tagged Hfq (3xFLAG hfq+). Resulting strains were exposed to 1O2 for 30 min at an OD660 of 0.4. Cell extracts were applied to total RNA (input) and coIP RNA (output) extraction. Six μg of total RNA and 350 ng of coIP RNA were loaded on polyacrylamide gels for detection of Pos19.
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pone.0163425.g005: Regulation of RSP_0557 by Pos19 depends on Hfq.(A) Results from in vivo reporter studies. Gene fragments containing the first codons and part of the upstream region were translationally fused to the lacZ gene on plasmid pPHU235. The corresponding reporter plasmids were transferred to control (pBBR1) and over-expression strains of Pos19 (pPos19) in wild-type (wt 2.4.1) and hfq deletion mutant (Δhfq) backgrounds. Cultures were stressed with 1O2 for 60 min and samples subjected to β-galactosidase assays. Bars indicate the relative β-galactosidase activity as calculated from Miller units (with the pBBR1 control set to 100% for each construct). Results represent the mean from three independent experiments with technical duplicates and error bars reflect the standard deviation. (B) Northern blot results from Hfq coIP experiments. Control plasmid pBBR1 and over-expression plasmid pPos19 were transferred to strains either expressing wild-type Hfq (wt hfq+) or 3xFLAG-tagged Hfq (3xFLAG hfq+). Resulting strains were exposed to 1O2 for 30 min at an OD660 of 0.4. Cell extracts were applied to total RNA (input) and coIP RNA (output) extraction. Six μg of total RNA and 350 ng of coIP RNA were loaded on polyacrylamide gels for detection of Pos19.

Mentions: To further elucidate the regulatory potential of Pos19, we made use of an in vivo reporter system, in which mRNAs are translationally fused to the promoter-less lacZ gene on reporter plasmid pPHU235 and transcribed from a 16S rRNA promoter [23]. On a second plasmid, Pos19 is over-expressed from its native RpoE-dependent promoter (Fig 3A). A fusion of takP [17] was used as negative control and, as expected, was not regulated by Pos19 in neither wild-type nor Δhfq background (Fig 5A). In addition to the microarray results, data from a target prediction by IntaRNA analysis [29,30] pointed to the possibility of RSP_0557 and cysH to be targets of Pos19 with interactions predicted for the translation initiation region, which we, therefore, tested in the reporter system. RSP_0557 expression was decreased to 32–37% in all tested Pos19 over-expression constructs (Fig 5A). These data support the observations from qRT-PCR experiments, where diminishment of mRNA levels was independent of an intact sORF. When the plasmid pPos19 was transferred to an hfq deletion mutant (Δhfq), the negative effect on the 0557-lacZ reporter was not observable (Fig 5A). Former results suggested that Pos19 is not an Hfq-binder [25] and the abolished function of Pos19 in the Δhfq background was therefore surprising. Assuming that Pos19 might weakly bind to Hfq and has not overcome the detection limit in the former study, co-immunoprecipitation (coIP) with 3xFLAG-tagged Hfq was repeated with a strain carrying pPos19, demonstrating that Pos19 clearly binds Hfq when present in elevated amounts (Fig 5B). Since the 0557-lacZ reporter carries parts of the RSP_0557 upstream region containing a potential promoter, an effect of Pos19 on the transcription of RSP_0557 could not be ruled out. The upstream region of RSP_0557 containing the potential promoter was transcriptionally fused to the lacZ gene containing its own ribosomal binding site. The activity of the RSP_0557 promoter did not differ in the empty vector control and Pos19 over-expression strain (S4 Fig).


Characteristics of Pos19 – A Small Coding RNA in the Oxidative Stress Response of Rhodobacter sphaeroides
Regulation of RSP_0557 by Pos19 depends on Hfq.(A) Results from in vivo reporter studies. Gene fragments containing the first codons and part of the upstream region were translationally fused to the lacZ gene on plasmid pPHU235. The corresponding reporter plasmids were transferred to control (pBBR1) and over-expression strains of Pos19 (pPos19) in wild-type (wt 2.4.1) and hfq deletion mutant (Δhfq) backgrounds. Cultures were stressed with 1O2 for 60 min and samples subjected to β-galactosidase assays. Bars indicate the relative β-galactosidase activity as calculated from Miller units (with the pBBR1 control set to 100% for each construct). Results represent the mean from three independent experiments with technical duplicates and error bars reflect the standard deviation. (B) Northern blot results from Hfq coIP experiments. Control plasmid pBBR1 and over-expression plasmid pPos19 were transferred to strains either expressing wild-type Hfq (wt hfq+) or 3xFLAG-tagged Hfq (3xFLAG hfq+). Resulting strains were exposed to 1O2 for 30 min at an OD660 of 0.4. Cell extracts were applied to total RNA (input) and coIP RNA (output) extraction. Six μg of total RNA and 350 ng of coIP RNA were loaded on polyacrylamide gels for detection of Pos19.
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pone.0163425.g005: Regulation of RSP_0557 by Pos19 depends on Hfq.(A) Results from in vivo reporter studies. Gene fragments containing the first codons and part of the upstream region were translationally fused to the lacZ gene on plasmid pPHU235. The corresponding reporter plasmids were transferred to control (pBBR1) and over-expression strains of Pos19 (pPos19) in wild-type (wt 2.4.1) and hfq deletion mutant (Δhfq) backgrounds. Cultures were stressed with 1O2 for 60 min and samples subjected to β-galactosidase assays. Bars indicate the relative β-galactosidase activity as calculated from Miller units (with the pBBR1 control set to 100% for each construct). Results represent the mean from three independent experiments with technical duplicates and error bars reflect the standard deviation. (B) Northern blot results from Hfq coIP experiments. Control plasmid pBBR1 and over-expression plasmid pPos19 were transferred to strains either expressing wild-type Hfq (wt hfq+) or 3xFLAG-tagged Hfq (3xFLAG hfq+). Resulting strains were exposed to 1O2 for 30 min at an OD660 of 0.4. Cell extracts were applied to total RNA (input) and coIP RNA (output) extraction. Six μg of total RNA and 350 ng of coIP RNA were loaded on polyacrylamide gels for detection of Pos19.
Mentions: To further elucidate the regulatory potential of Pos19, we made use of an in vivo reporter system, in which mRNAs are translationally fused to the promoter-less lacZ gene on reporter plasmid pPHU235 and transcribed from a 16S rRNA promoter [23]. On a second plasmid, Pos19 is over-expressed from its native RpoE-dependent promoter (Fig 3A). A fusion of takP [17] was used as negative control and, as expected, was not regulated by Pos19 in neither wild-type nor Δhfq background (Fig 5A). In addition to the microarray results, data from a target prediction by IntaRNA analysis [29,30] pointed to the possibility of RSP_0557 and cysH to be targets of Pos19 with interactions predicted for the translation initiation region, which we, therefore, tested in the reporter system. RSP_0557 expression was decreased to 32–37% in all tested Pos19 over-expression constructs (Fig 5A). These data support the observations from qRT-PCR experiments, where diminishment of mRNA levels was independent of an intact sORF. When the plasmid pPos19 was transferred to an hfq deletion mutant (Δhfq), the negative effect on the 0557-lacZ reporter was not observable (Fig 5A). Former results suggested that Pos19 is not an Hfq-binder [25] and the abolished function of Pos19 in the Δhfq background was therefore surprising. Assuming that Pos19 might weakly bind to Hfq and has not overcome the detection limit in the former study, co-immunoprecipitation (coIP) with 3xFLAG-tagged Hfq was repeated with a strain carrying pPos19, demonstrating that Pos19 clearly binds Hfq when present in elevated amounts (Fig 5B). Since the 0557-lacZ reporter carries parts of the RSP_0557 upstream region containing a potential promoter, an effect of Pos19 on the transcription of RSP_0557 could not be ruled out. The upstream region of RSP_0557 containing the potential promoter was transcriptionally fused to the lacZ gene containing its own ribosomal binding site. The activity of the RSP_0557 promoter did not differ in the empty vector control and Pos19 over-expression strain (S4 Fig).

View Article: PubMed Central - PubMed

ABSTRACT

The phototrophic bacterium Rhodobacter sphaeroides induces several small RNAs (sRNAs) when singlet oxygen (1O2) levels are elevated, a situation also referred to as photo-oxidative stress. An RNA-seq study identified the RSs0019 sRNA, which is renamed Pos19 (photo-oxidative stress induced sRNA 19). Pos19 is part of the RpoE regulon and consequently induced upon 1O2 and peroxide stress. The 219 nt long Pos19 transcript contains a small open reading frame (sORF) of 150 nt, which is translated in vivo. Over-expression of Pos19 results in reduced mRNA levels for several genes, of which numerous are involved in sulfur metabolism. The negative effect on the potential targets is maintained even when translation of the sORF is abolished, arguing that regulation is entailed by the sRNA itself. Reporter studies further revealed that regulation of the most affected mRNA, namely RSP_0557, by Pos19 is Hfq-dependent. Direct binding of Pos19 to Hfq was shown by co-immunoprecipitation. Physiological experiments indicated Pos19 to be involved in the balance of glutathione biosynthesis. Moreover, a lack of Pos19 leads to elevated reactive oxygen species levels. Taken together our data identify the sRNA Pos19 as a coding sRNA with a distinct expression pattern and potential role under oxidative stress in the phototrophic bacterium R. sphaeroides.

No MeSH data available.